The proposed research is designed to advance our basic knowledge and understanding of gene expression in higher organisms. A significant objective is to elucidate the nature of the complex control mechanisms which regulate the major pathways of nitrogen and sulfur metabolism and to look for possible interactions between these distinct control circuits. Efficient techniques will be employed to isolate specific types of regulatory mutants in Neurospora. These mutants will be studied by parallel genetic and biochemical approaches to reveal their mode of action. The complex control by multiple circuits of the structural gene which encodes an extracellular protease will be similarly studied with an emphasis on the extensive control region predicted to occur adjacent to the structural gene. The various structural and regulatory gene mutants (and the mechanism of regulation which they imply) will be utilized as a basis to study regulation at the molecular level. Messenger RNA species for specific nitrogen-controlled and sulfur-controlled enzymes will be purified by immunoprecipitation of the respective polysome fraction. Complementary DNA "probes", prepared from the specific mRNAs with reverse transcriptase, will then be employed to study transcriptional control of gene expression both in vivo and in vitro. It is planned to devise an in vitro transcriptional system in which isolated chromatin will serve as the template for RNA synthesis. Chromatin reconstitution studies will be utilized to detect positive-acting regulatory gene products, predicted to be nuclear proteins, which will be subsequently purified and studied. The long term goal of the proposed research is to elucidate at the molecular level, the regulatory elements and the complex mechanisms, which control gene expression in eukaryotes.